v Abstract Dental caries is a multifactorial disease that results from the interaction between the dental biofilm, oral environment and tooth structure of an individual. Although cross-sectional studies have suggested some species are associated more commonly with carious sites and others with caries-free surfaces,3, 4 the presence or absence of a specific species has not been shown to be predictive of future caries development or caries progression. New technological advances have led to the understanding of the molecular mechanisms of many diseases, but despite our knowledge of the basic concepts of dental caries, the interaction of the dental biofilm microbiota, which may serve as an important caries risk indicator,1-4 is not well understood. We propose to analyze dental biofilm metabolites in relation to the caries status of the surface associated with the biofilm. Our goal is to understand the metabolic activity of the dental biofilm to ultimately identify a signature for caries activity. Our current grant (NIH 5R01DE017890-05), has detailed data on over 300 caries active children, well characterized by the International Caries Detection and Assessment System (ICDAS) and by Quantitative Light Induced Fluorescence (QLF) longitudinally in terms of their caries activity and caries risk for the past four years. This allows us a unique window of opportunity to identify if there is a potential signature associated with dental caries by selecting children and specific tooth surfaces that are either caries-free (C-F), or caries-active (C-A) (have progressing lesions). The hypothesis for this study is that caries lesion activity is a reflection of the activity of the overlaying dental biofilm; therefore, active lesions can be distinguished from arrested lesions and sound surfaces based upon their metabolic activity. Thus, we propose, to select and longitudinally examine and collect biofilm of 450 children (300 C-A and 150 C-F) between the ages of 5-19 to determine: (1) if there are marked differences between dental biofilm metabolic activities using gas chromatography - mass spectrometry (GC-MS) on a site-specific analysis that may categorize C-F surfaces compared to surfaces with C-A lesions in C-A children; (2) if there is a distinction between the metabolite signals in the biofilm overlaying sound surfaces from C-F children compared to C-A children; and (3) if there is a shift in the metabolite signals of the dental biofilm overlaying surfaces that progress to cavitation. Pinpointing the metabolite signals associated with sound and C-A surfaces and with caries progression will allow the identification of biomarker(s) for caries activity that can be developed in a caries diagnostic test with unprecedented prediction power. This work is being done is close collaboration with experts in bioanalytical chemistry, specifically metabolomics and organic volatile compounds. Our research team now includes Drs. Soini and Novotny from the IU Department of Chemistry, and Institute for Pheromone Research as well as current collaborators in the University of Puerto Rico, where our study population is located.